Antibacterial Components and Modes of the Methanol-Phase Extract from Commelina communis Linn

Infectious diseases caused by pathogenic bacteria severely threaten human health. Traditional Chinese herbs are potential sources of new or alternative medicine. In this study, we analyzed for the first time antibacterial substances in the methanol-phase extract from a traditional Chinese herb—Commelina communis Linn—which showed an inhibition rate of 58.33% against 24 species of common pathogenic bacteria. The extract was further purified using preparative high-performance liquid chromatography (Prep-HPLC), which generated four single fragments (Fragments 1 to 4). The results revealed that Fragment 1 significantly increased bacterial cell surface hydrophobicity and membrane permeability and decreased membrane fluidity, showing disruptive effects on cell integrity of Gram-positive and Gram-negative bacteria, such as Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus, and Salmonella enterica subsp., compared to the control groups (p < 0.05). In sum, 65 compounds with known functions in Fragment 1 were identified using liquid chromatography and mass spectrometry (LC-MS), of which quercetin-3-o-glucuronide was predominant (19.35%). Comparative transcriptomic analysis revealed multiple altered metabolic pathways mediated by Fragment 1, such as inhibited ABC transporters, ribosome, citrate cycle and oxidative phosphorylation, and upregulated nitrogen metabolism and purine metabolism, thereby resulting in the repressed bacterial growth and even death (p < 0.05). Overall, the results of this study demonstrate that Fragment 1 from C. communis Linn is a promising candidate against common pathogenic bacteria.


Introduction
Commelina communis Linn (known as herba commelinae) is an annual herb that grows in tropical and subtropical regions of the world [1]. This plant belongs to the phylum of Angiospermae, class Monocotyledoneae, suborder Commelinineae, family Commelinaceae, and genus Commelina. C. communis Linn is often used as a febrifuge or diuretic in Chinese folk medicine to treat high fever, sore throat, edema oliguria, astringent pain, and bloated furuncle poison [2]. The herb was recorded in the Pharmacopoeia of the People's Republic of China in 1977. Recent research has also shown that C. communis Linn contains alpha-glucosidase-inhibiting polyhydroxyalkaloids and could be used to therapy noninsulin-dependent diabetes [3]. Nevertheless, current literature on bacteriostatic activity of C. communis Linn is rare. To the best of our knowledge, only Tang and colleagues [4] reported that the ethyl acetate extract of C. communis Linn contained effective antibacterial components, and its minimum inhibitory concentrations (MICs) against Staphylococcus aureus, Staphylococcus albus, Escherichia coli, and Salmonella typhi were 10 mg/mL. They also identified bioactive compounds n-triacontanol, D-mannitol, p-hydroxycinnamic acid, and daucosteril, the latter two of which showed antibacterial activity and antitussive effect, respectively [4]. However, the underlying molecular mechanisms remain unidentified.
Medicinal plants are a very good source for obtaining a variety of bioactive compounds and drugs [5]. For example, recently, Abed et al. [6] reported the phytochemical composition, and antibacterial, antioxidant, and antidiabetic potential of methanolic extracts of Cydonia oblonga bark [6]. Al-Joufi et al. [7] found that Anabasis articulata (Forssk.) Moq is a good source of phytochemicals with antibacterial, antioxidant, and antidiabetic potential [7].
To further exploit bioactive nature products in C. communis Linn, in this study, we used the methanol and chloroform extraction method, established recently in our research groups [8][9][10], to extract bacteriostatic components from C. communis Linn and investigate possible antibacterial mechanisms. The objectives of this study were: (1) to determine inhibition activity of methanol-phase and chloroform-phase crude extracts from C. communis Linn against 24 species of common pathogenic bacteria; (2) to purify the methanol-phase extract from C. communis Linn using preparative high-performance liquid chromatography (Prep-HPLC) and identify antibacterial compounds using liquid chromatography mass spectrometry (LC-MS); (3) to monitor cell structure changes of four representative bacterial strains mediated by Fragment 1 of the methanol-phase extract; and (4) to decipher molecular mechanisms underlying the antibacterial activity mediated by Fragment 1 by comparative transcriptomic analysis. The results of this study provide useful data for potential pharmaceutical application of C. communis Linn against common pathogenic bacteria.

Antibacterial Activity of Methanol-and Chloroform-Phase Crude Extracts from C. communis Linn
Antibacterial substances in fresh leaf and stem tissues of C. communis Linn were extracted using the methanol and chloroform extraction method (see the Materials and Methods). We found that the water loss rate of the fresh tissues was 89.57% after freezedrying treatment of the fresh samples. The observed extraction yields of the methanol-phase and chloroform-phase crude extracts were 30.50% and 16.50%, respectively.
The inhibition rates of the crude extracts against 24 species of common pathogenic bacteria were determined, and the results are shown in Table 1. The chloroform-phase extract from C. communis Linn had a bacteriostatic rate of 50.00%, and inhibited two species of Gram-positive bacteria, S. aureus and Enterococcus faecalis, and 10 species of Gramnegative bacteria: Enterobacter cloacae, E. coli, Salmonella paratyphi-A (ex Kauffmannand Edwards), Salmonella, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio mimicus ( Table 1).
Methanol and chloroform are organic regents that have been used for effective extraction of bioactive compounds from different pharmacophagous plants [9,10]. The results of this study provided additional evidence of the extraction method for more effectively isolating antibacterial substances from C. communis Linn compared to ethyl acetate, with which only four species of bacteria were inhibited: S. aureus, S. albus, E. coli, and S. typhi [7].
In this study, the MICs of the two extractions were determined: 32-1024 µg/mL for the methanol-phase extract and 64-1024 µg/mL for the chloroform-phase extract ( Table 1). The MIC values of the two extracts were much lower than that of the ethyl acetate extract (10 mg/mL) [7], indicating the higher extraction efficiency of the method used in this study.
Given the higher inhibition rate (58.33%) of the methanol-phase extract from C. communis Linn, this crude extract was chosen for further analysis in this study. ----Note: CPE: chloroform-phase extract. MPE: methanol-phase extract. -: no bacteriostasis activ Inhibition zone: diameter includes the disk diameter (6 mm). MIC: minimum inhibitory concent tion. Values presented as means ± standard deviation (S.D.) of three parallel measurements.

Purification of the Methanol-Phase Crude Extract from C. communis Linn
The methanol-phase crude extract from C. communis Linn was prepared on a lar scale and subjected to Prep-HPLC analysis. This analysis revealed four obviously sep rated fragments (designated Fragments 1 to 4) by scanning at OD220 for 12 min ( Figure S These four single fragments were individually collected, and their antibacterial tivities were determined ( Table 2). The results showed that Fragment 1 highly inhibit the growth of S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Pop serovar Vellore ATCC15611, S. aureus ATCC25923, B. cereus A1-1, and E. faecalis C1-1 co pared to the control groups (p < 0.05). Moreover, the growth of S. aureus ATCC8095, alginolyticus ATCC17749, V. metschnikovii ATCC700040, and V. parahaemolyticus B5-29 w also significantly repressed (p < 0.05). Among these, S. enterica subsp. enterica (ex Kau mann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611 is a Gram-negat pathogen that can cause mild, self-limiting enterocolitis to systemic (typhoid) diseases humans [11]. E. faecalis is an opportunistic pathogen involved in severe hospital-acquir infections [12]. S. aureus can cause pneumonia, endocarditis, and bacteremia upon gaini access to the bloodstream of the host [13]. B. cereus can lead to food poisoning and can a cause gastrointestinal disorders and severe systemic infections in humans [14].
In contrast, weak or no antibacterial activity was observed from the other three fra ments (Fragments 2 to 4), indicating that the majority of antibacterial compounds exist in Fragment 1 of the methanol-phase extract from C. communis Linn.
The MIC values of Fragment 1 were also determined, and the results are present in Table 2. For example, S. enterica ATCC15611 was strongly inhibited by Fragment 1 fro C. communis Linn. at 128 μg/mL, with S. aureus ATCC25923 at 256 μg/mL, and B. cer A1-1 and E. faecalis C1-1 at 512 μg/mL, respectively.

Purification of the Methanol-Phase Crude Extract from C. communis Linn
The methanol-phase crude extract from C. communis Linn was prepared on a large scale and subjected to Prep-HPLC analysis. This analysis revealed four obviously separated fragments (designated Fragments 1 to 4) by scanning at OD 220 for 12 min ( Figure S1).
These four single fragments were individually collected, and their antibacterial activities were determined ( Table 2). The results showed that Fragment 1 highly inhibited the growth of S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611, S. aureus ATCC25923, B. cereus A1-1, and E. faecalis C1-1 compared to the control groups (p < 0.05). Moreover, the growth of S. aureus ATCC8095, V. alginolyticus ATCC17749, V. metschnikovii ATCC700040, and V. parahaemolyticus B5-29 was also significantly repressed (p < 0.05). Among these, S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611 is a Gram-negative pathogen that can cause mild, self-limiting enterocolitis to systemic (typhoid) diseases in humans [11]. E. faecalis is an opportunistic pathogen involved in severe hospital-acquired infections [12]. S. aureus can cause pneumonia, endocarditis, and bacteremia upon gaining access to the bloodstream of the host [13]. B. cereus can lead to food poisoning and can also cause gastrointestinal disorders and severe systemic infections in humans [14]. In contrast, weak or no antibacterial activity was observed from the other three fragments (Fragments 2 to 4), indicating that the majority of antibacterial compounds existed in Fragment 1 of the methanol-phase extract from C. communis Linn.
The MIC values of Fragment 1 were also determined, and the results are presented in Table 2. For example, S. enterica ATCC15611 was strongly inhibited by Fragment 1 from C. communis Linn. at 128 µg/mL, with S. aureus ATCC25923 at 256 µg/mL, and B. cereus A1-1 and E. faecalis C1-1 at 512 µg/mL, respectively.

Effects of Fragment 1 from C. communis Linn on Bacterial Cell Structure
Bacterial cell surface hydrophobicity determines the ability of bacteria to adhere to inert surfaces, which plays a key role in bacterial colonization in the host [15]. In this study, after treatment with Fragment 1 from C. communis Linn for 2 h, cell surface hydrophobicity of S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611, S. aureus ATCC25923, E. faecalis C1-1 and B. cereus A1-1 was significantly increased by 1.53-fold to 2.13-fold compared to the control groups (p < 0.05) (Figure 2A). After being treated for 4 h, higher cell surface hydrophobicity was observed (1.89-fold to 2.47-fold). The highest increase (3.06-fold) was observed in the S. enterica ATCC15611 treatment group after being treated for 6 h (Figure 2A). Based on the above results, we further examined bacterial inner cell membrane permeability using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a probe, and the results are illustrated in Figure 3. For example, the inner cell membrane permeability of S. enterica ATCC15611 increased significantly after treatment with Fragment 1 for 2 h (1.07-fold), 4 h (1.08-fold), and 6 h (1.11-fold), respectively ( Figure 3A). For B. cereus A1-1, its inner cell Cell membrane fluidity is also a key property for maintaining the viability of cells and intracellular metabolic functions, particularly under stress [16]. Changes in membrane fluidity have concomitant effects on membrane protein activities and intercellular communication [17]. In this study, the four treatment groups showed significant changes in cell membrane fluidity compared to the control groups (p < 0.05) ( Figure 2B). For example, after being treated with Fragment 1 from C. communis Linn for 2 h, the cell membrane fluidity of S. enterica ATCC15611, S. aureus ATCC25923, E. faecalis C1-1 and B. cereus A1-1 was significantly decreased by 2.10-fold, 1.07-fold, 1.04-fold and 1.28-fold, respectively (p < 0.05). Upon prolonged treatment for 4 h and 6 h, among the four strains, the most significant decrease in cell membrane fluidity was observed in the S. enterica ATCC15611 treatment group (4.80-fold and 5.66-fold) ( Figure 2B). The change in membrane lipid composition may contribute to the observed membrane fluidity change by the therapeutic agents [18].
As shown in Figure 2C, cell membrane damage rates were significantly increased in the four treatment groups compared to the control groups (p < 0.05). After being treated with Fragment 1 for 2 h, significantly increased damage rates were observed in B. cereus A1-1 (3.42-fold), S. aureus ATCC25923 (3.36-fold), S. enterica ATCC15611 (2.88-fold), and E. faecalis C1-1 (1.45-fold) treatment groups (p < 0.05). The damage was aggravated after four hours of the treatment, and the most serious damage was observed in S. enterica ATCC15611 (6.64-fold), whereas the damage rate of E. faecalis C1-1 was the lowest (3.12-fold). The cell membrane damage of S. enterica ATCC15611 was also the most severe among the four stains after being treated with Fragment 1 for 6 h (8.52-fold) ( Figure 2C).
Based on the above results, we further examined bacterial inner cell membrane permeability using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a probe, and the results are illustrated in Figure 3. For example, the inner cell membrane permeability of S. enterica ATCC15611 increased significantly after treatment with Fragment 1 for 2 h (1.07-fold), 4 h (1.08-fold), and 6 h (1.11-fold), respectively ( Figure 3A). For B. cereus A1-1, its inner cell membrane permeability did not change significantly after the treatment for 2 h (p > 0.05), but increased by 1.12-fold and 1.13-fold after the treatment for 4 h, and 6 h, respectively (p < 0.05) ( Figure 3B). Similarly, E. faecalis C1-1 did not show a significant change in inner membrane permeability within 2 h of the treatment, but was significantly increased after 4 h (1.05-fold) and 6 h (1.05-fold) (p < 0.05) ( Figure 3C). S. aureus ATCC25923 had no significant change in inner cell membrane permeability after being treated for 2 h and 4 h, but showed significantly increased permeability after treatment for 6 h (1.08-fold) ( Figure 3D). These results indicated that Fragment 1 from C. communis Linn displays different damage effects on inner cell membrane permeability of the four bacterial strains.
We also examined bacterial outer membrane permeability using N-Phenyl-1-naphthylamine (NPN) as a probe, and the results are presented in Figure 4. The outer membrane permeability in the four treatment groups was all significantly increased (1.17-fold to 3.52-fold) after the treatment with Fragment 1 for 2 h (p < 0.05). A higher increase in outer membrane permeability was observed in B. cereus A1-1 (4.36-fold) and S. enterica ATCC15611 (2.38-fold) groups after being treated for 4 h (p < 0.001), followed by E. faecalis C1-1 (2.00-fold), and S. aureus ATCC25923 (1.39-fold) (p < 0.01). After 6 h of the treatment, the outer membrane permeability of B. cereus A1-1 also increased most significantly (5.07-fold), while that of S. aureus ATCC25923 was the opposite (1.34-fold).
In conclusion, these results indicated that Fragment 1 from C. communis Linn can affect cell membrane structures of S. enterica ATCC15611, E. faecalis C1-1, S. aureus ATCC25923 and B. cereus A1-1, particularly increasing cell surface hydrophobicity and membrane permeability, but reducing membrane fluidity, and significantly damage cell membrane structures, in agreement with bacterial surface structure changes observed by scanning electron microscopy (SEM) analysis (see below). The damage to cell membranes was conducive to the penetration of fragment 1 of C. communis Linn into bacterial cells, consequently influencing intracellular metabolisms and thereby inhibiting bacterial growth and even leading to bacterial death.  In conclusion, these results indicated that Fragment 1 from C. communis Linn can affect cell membrane structures of S. enterica ATCC15611, E. faecalis C1-1, S. aureus ATCC25923 and B. cereus A1-1, particularly increasing cell surface hydrophobicity and membrane permeability, but reducing membrane fluidity, and significantly damage cell membrane structures, in agreement with bacterial surface structure changes observed by scanning electron microscopy (SEM) analysis (see below). The damage to cell membranes was conducive to the penetration of fragment 1 of C. communis Linn into bacterial cells, consequently influencing intracellular metabolisms and thereby inhibiting bacterial growth and even leading to bacterial death.

Bacterial Cell Surface Structure Changes Observed by the SEM Assay
Based on the above results, we further observed bacterial cell surface structure changes by the SEM assay ( Figure 5). In the control groups, cells of coccus were spherical or slightly elliptical with smooth surface and clear structure, while cells of bacillus were rods, round at both ends, smooth and complete in surface. For example, B. cereus A1-1 was a rod-shaped Gram-positive bacterium. The bacterial cells in the control group were intact

Bacterial Cell Surface Structure Changes Observed by the SEM Assay
Based on the above results, we further observed bacterial cell surface structure changes by the SEM assay ( Figure 5). In the control groups, cells of coccus were spherical or slightly elliptical with smooth surface and clear structure, while cells of bacillus were rods, round at both ends, smooth and complete in surface. For example, B. cereus A1-1 was a rod-shaped Gram-positive bacterium. The bacterial cells in the control group were intact in shape with visible pili. However, after the treatment with Fragment 1 from C. communis Linn for 2 h, the cell surface shrank. The change showed a treatment time-dependent mode, and a large number of the bacterial cells broke, and cellular contents leaked after the treatment for 6 h.

Identification of Potential Antibacterial Compounds in Fragment 1 from C. communis Linn
In order to identify antibacterial compounds in C. communis Linn, Fragment 1 was further subjected to UHPLC-MS analysis. Approximately 65 compounds with known functions were identified, the highest percentage of which was quercetin-3-o-glucuronide (19.35%), followed by glutamine (8.69%), sucrose (6.46%), methyl gallate (4.93%), and indole (4.52%) ( Table 3). The major compound classes included flavonoids, alkaloids, phenols, terpenoids, and steroids. Studies have indicated that the flavonoid quercetin-3-oglucuronide has anti-inflammatory, antiviral, and antiallergic properties [19,20]. Quercetin-3-o-glucuronide is a pharmacologically active flavonol glucuronide, and Kawai recently found unique actions at sites of inflammation, including specific accumulation in macrophages and the following deconjugation into active aglycone, catalyzed by the macrophage-derived β-glucuronidase [21]. Alkaloids exhibit various biological functions such as antitumor, antiviral, antimicrobial and anti-inflammatory activities [22]. Terpenoids, such as kaurenoic acid, miltirone, kirenol, and shionone, identified in this study, are the largest class of natural products, most of which are derived from plants [23]. They play important roles in food and pharmaceutical fields due to diverse biological and pharmacological activities [24]. Terpenoids have huge potential against microorganisms through different mechanisms, such as membrane disruption, anti-quorum sensing, and inhibition of protein synthesis [25]. The combination therapy of terpenoids and antimicrobial agents has increased the potency of treatment against multidrug-resistant microorganisms by showing synergism to each other [25]. For example, methyl gallate, identified in this study, can inhibit bacterial virulence and reduce membrane integrity and cell survival rate of S. typhimurium [26]. Additionally, trigonelline, identified in this study, has antibacterial, antiviral and antitumor activities, with therapeutic potential for diabetes and central nervous system disease [27].  Similarly, for the Gram-positive S. aureus ATCC25923, in the control group, the surface of the bacterial cells was clear, smooth and wrinkle-free with the typical spherical structure. However, after being treated with Fragment 1 for 2 h, although most cells were plump and spherical with complete cell structure, a small number of the cells shrank on the surface. After treatment for 4 h, the surface of most cells showed slight wrinkles, and even collapsed and lost the spherical structure after the treatment for 6 h.
For the Gram-positive E. faecalis C1-1, the control group cells were single, paired or short chains of ovoid cocci with smooth surface and intact morphological structure. No obvious change was observed after treatment with Fragment 1 for 2 h. However, after being treated for 4 h, a large number of the bacterial cells had sunk on the surface, and the cells severely shrank and deformed. After the treatment for 6 h, a large amount of cellular content leaked and the bacterial cells lost the spherical structure.
For the Gram-negative S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611, the control group was morphologically intact with a short rod shape. It did not change significantly after treatment with Fragment 1 for 2 h. However, after being treated for 4 h, the cell surface slightly contracted, and some cells broke. The bacterial cells shrank severely after treatment for 6 h.
These results demonstrated that Fragment 1 from C. communis Linn (1 MIC) can cause varying degrees of damage on cellular surface structure of both Gram-positive and Gramnegative bacteria, especially the coccus, which showed the most obvious shrinkage, fracture or cavities on the cell surface.

Identification of Potential Antibacterial Compounds in Fragment 1 from C. communis Linn
In order to identify antibacterial compounds in C. communis Linn, Fragment 1 was further subjected to UHPLC-MS analysis. Approximately 65 compounds with known functions were identified, the highest percentage of which was quercetin-3-o-glucuronide (19.35%), followed by glutamine (8.69%), sucrose (6.46%), methyl gallate (4.93%), and indole (4.52%) ( Table 3). The major compound classes included flavonoids, alkaloids, phenols, terpenoids, and steroids. Studies have indicated that the flavonoid quercetin-3-oglucuronide has anti-inflammatory, antiviral, and antiallergic properties [19,20]. Quercetin-3o-glucuronide is a pharmacologically active flavonol glucuronide, and Kawai recently found unique actions at sites of inflammation, including specific accumulation in macrophages and the following deconjugation into active aglycone, catalyzed by the macrophage-derived β-glucuronidase [21].  Alkaloids exhibit various biological functions such as antitumor, antiviral, antimicrobial and anti-inflammatory activities [22]. Terpenoids, such as kaurenoic acid, miltirone, kirenol, and shionone, identified in this study, are the largest class of natural products, most of which are derived from plants [23]. They play important roles in food and pharmaceutical fields due to diverse biological and pharmacological activities [24]. Terpenoids have huge potential against microorganisms through different mechanisms, such as membrane disruption, anti-quorum sensing, and inhibition of protein synthesis [25]. The combination therapy of terpenoids and antimicrobial agents has increased the potency of treatment against multidrug-resistant microorganisms by showing synergism to each other [25]. For example, methyl gallate, identified in this study, can inhibit bacterial virulence and reduce membrane integrity and cell survival rate of S. typhimurium [26]. Additionally, trigonelline, identified in this study, has antibacterial, antiviral and antitumor activities, with therapeutic potential for diabetes and central nervous system disease [27].

Differential Transcriptomes Mediated by Fragment 1 from C. communis Linn
To obtain further insights into the genome-wide gene expression changes mediated by Fragment 1 from C. communis Linn, we determined transcriptomes of S. enterica subsp. Approximately 11.05% (506/4578) of S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611 genes were expressed differently in the treatment group compared to the control group. Among these, 246 DEGs showed higher transcription levels (FC ≥ 2.0), whereas 260 DEGs were downregulated (FC ≤ 0.5) (p < 0.05). Approximately 12 significantly changed metabolic pathways were identified, including the ribosome; citrate cycle; glycolysis/gluconeogenesis; oxidative phosphorylation; carbon fixation pathways in prokaryotes; RNA degradation; purine metabolism; methane metabolism; alanine, aspartate and glutamate metabolism; nitrogen metabolism; pyruvate metabolism; and galactose metabolism ( Figure 6, Table 4).    Of note, approximately 68 DEGs involved in 12 changed metabolic pathways were significantly downregulated at the transcription level in S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611 (0.009-fold to 0.500-fold) (p < 0.05) ( Table 4). For example, in the citrate cycle, the expression of 10 DEGs was significantly inhibited (0.084-fold to 0.500-fold) (p < 0.05). Remarkably, the DEGs encod-ing a fumarate hydratase (FH) class I aerobic (SPC_2263) and a succinate dehydrogenase catalytic subunit (SPC_0731) were greatly downregulated (0.084-fold and 0.097-fold). The FH catalyzes the conversion of fumarate to L-malate as part of the tricarboxylic acid cycle (TCA) [28]. Fumarate has been shown to inhibit α-ketoglutarate-dependent dioxygenases that are involved in DNA and histone demethylation [29]. In this study, five DEGs encoding key enzymes in the oxidative phosphorylation were also significantly repressed (0.057-fold to 0.497-fold) (p < 0.05), including a succinate dehydrogenase cytochrome b556 small membrane subunit (SPC_0730), a NADH dehydrogenase alpha subunit (SPC_1381), a NADH dehydrogenase I chain 2CD (SPC_1383), and a NADH dehydrogenase subunit H (SPC_1387), and an inorganic pyrophosphatase (SPC_4565). Additionally, in the carbon fixation pathways in prokaryotes, the DGEs encoding an acetyl-coenzyme synthetase (SPC_4339) and a succinate dehydrogenase catalytic subunit (SPC_0732) were significantly downregulated as well (0.047-fold and 0.127-fold) (p < 0.05). Succinate dehydrogenase is a heterotetrameric protein complex that links the TCA with the electron transport chain [30]. In the methane metabolism, the expression of three DEGs was also significantly repressed (0.225-fold to 0.434-fold) (p < 0.05), including a phosphate acetyltransferase (SPC_1369), a phosphoenolpyruvate synthase (SPC_2381), and a formate dehydrogenase-O gamma subunit (SPC_4138). The above five metabolic pathways were involved in energy metabolism, the significantly downregulation of which indicated that the energy supply is deficient in S. enterica ATCC15611 mediated by Fragment 1 from C. communis Linn.
The comparative transcriptomic data demonstrated that Fragment 1 from C. communis Linn can significantly alter 12 metabolic pathways in S. enterica subsp. enterica (ex Kauffmann and Edwards) Le Minor and Popoff serovar Vellore ATCC15611, which results in inhibited carbohydrate metabolism, insufficient energy supply, and downregulated protein translation, and consequently affects bacterial survival.  Table 5).  Notably, approximately 71 DEGs involved in the seven changed metabolic pathways were significantly downregulated (0.012-fold to 0.494-fold) (p < 0.05) ( Table 5). For example, in oxidative phosphorylation, the expression of five DEGs was significantly inhibited    Notably, approximately 71 DEGs involved in the seven changed metabolic pathways were significantly downregulated (0.012-fold to 0.494-fold) (p < 0.05) ( Table 5). For example, in oxidative phosphorylation, the expression of five DEGs was significantly inhibited at the transcription level (0.249-fold to 0.350-fold) (p < 0.05), including an ATP synthase F1 epsilon subunit (SAOUHSC_02340), a quinol oxidase subunit IV putative (SAOUHSC_00999), a cytochrome c oxidase subunit III putative (SAOUHSC_01000), a quinol oxidase subunit I (SAOUHSC_01001), and a quinol oxidase AA3 subunit II putative (SAOUHSC_01002). This metabolic pathway was related to energy metabolism, the downregulation of which indicated insufficient energy supply in S. aureus ATCC25923.
Comparative transcriptomic analysis also revealed that 24 DEGs encoding ABC transporters were significantly downregulated in S. aureus ATCC25923 (0.012-fold to 0.483-fold) (p < 0.05). ABC transporters are also known as efflux pumps that mediate the crossmembrane transportation of various endo-and xenobiotic molecules energized by ATP hydrolysis [41]. Therefore, ABC transporters have been considered closely in multidrug resistance (MDR), where the efflux of structurally distinct chemotherapeutic drugs causes reduced therapeutic efficacy [42]. In this study, the downregulated expression of these DEGs indicated that Fragment 1 from C. communis Linn can repress the pumping out of harmful substances by S. aureus ATCC25923 in order to eliminate cell damage.
Similarly to S. aureus ATCC25923, the expression of 51 DEGs in ABC transporters was significantly downregulated in B. cereus A1-1 (0.022-fold to 0.438-fold) (p < 0.05). Notably, the DEG encoding a cobalt transport protein (BCN_2523) was highly downregulated (0.030fold), which belongs to a family of secondary metal transporters in prokaryotes and fungi, characterized by an eight-transmembrane-domain architecture and mediating high-affinity uptake of cobalt and/or nickel ions into cells [51].
Comparative transcriptomic analysis indicated that Fragment 1 from C. communis Linn. can increase nitrogen metabolism and purine metabolism in B. cereus A1-1, but inhibits ABC transporters, valine, leucine, and isoleucine biosynthesis, cysteine and methionine metabolism, propanoate metabolism, sulfur metabolism, C5-branched dibasic acid metabolism, butanoate metabolism, citrate cycle, and beta-lactam resistance, which likely results in repressed energy metabolism, transport system, carbohydrate metabolism, protein synthesis, and drug resistance, and consequently influences cell growth and proliferation of B. cereus A1-1. The comparative transcriptomic analysis revealed six significantly changed metabolic pathways in E. faecalis C1-1: the ribosome; phenylalanine, tyrosine, and tryptophan biosynthesis; carbon fixation pathways in prokaryotes; fatty acid biosynthesis; thiamine metabolism; and ABC transporters ( Figure 9, Table 7).  The expression of a large number of DEGs (n = 52) encoding ABC transporters was significantly depressed in E. faecalis C1-1 (0.002-fold to 0.472-fold) (p < 0.05). Notably, the DEG encoding the substrate-binding protein (SBP) (IUJ47_RS12795) was extremely     The expression of a large number of DEGs (n = 52) encoding ABC transporters was significantly depressed in E. faecalis C1-1 (0.002-fold to 0.472-fold) (p < 0.05). Notably, the DEG encoding the substrate-binding protein (SBP) (IUJ47_RS12795) was extremely downregulated (0.002-fold), which is a key determinant of substrate specificity and high affinity of ABC uptake systems in bacteria and archaea. Most prokaryotes have many SBP-dependent ABC transporters that recognize a broad range of ligands from metal ions to amino acids, sugars and peptides [52].
For instance, Fragment 1 from C. communs Linn. affected the propanoate metabolism, sulfur metabolism, cysteine and methionine metabolism, C5-branched dibasic acid metabolism, butanoate metabolism, and beta-lactam resistance in only B. cereus A1-1, whereas arginine biosynthesis, PPAR signaling pathway, and carotenoid biosynthesis were repressed in S. aureus ATCC25923. These results demonstrated that Fragment 1 from C. communs Linn. can repress energy metabolism in the four tested strains, hinder signal transmission, reduce the pumping capacity of exogenous harmful substances, thereby inhibiting bacterial growth and even leading to cell death.

Bacterial Strains and Culture Conditions
The bacterial strains and culture media used in this study are listed in Table S3. Each of the strains was inoculated into the corresponding media supplemented with 3.0% NaCl (pH 8.4-8.5, Vibrio strains) or 0.5/1.0% NaCl (pH 7.0-7.2, non-Vibrio strains), respectively, and incubated at 37 • C, as described in our previous reports [9,10]. Luria-Bertani (LB) and tryptic soy broth (TSB) media were purchased from Beijing Land Bridge Technology Co., Ltd., Beijing, China. Brain heart infusion (BHI) and Marine 2216 media were purchased from Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China, and Becton, Dickinson and Company, New York, NY, USA, respectively.

Extraction of Bioactive Substances from C. communis Linn
The pharmacophagous plant C. communis Linn was purchased from the Caoxuan Food Store in Lishui City (27 • 25 37" N, 118 • 41 28" E) in Zhejiang Province, China in August 2020. The extraction of bioactive substances from C. communis Linn was carried out using the methanol and chloroform method described in our recent studies [9,10]. Briefly, an aliquot of 500 g fresh leaves and stems of C. communis Linn was freeze-dried at −80 • C for 48 h using the ALPHA 2-4 LD Plus Freeze-Dryer (Martin Christ, Osterode, Germany). The freeze-dried sample (10 g) was crushed, and then mixed with 99 mL chloroform:methanol (2:1, v/v) for 6 h, then subjected to ultrasonication using the Scientz IID ULtrasonic Cell Crusher (SCIENT Z, Ningbo, China) with the same parameters described previously [9]. The methanol phase and chloroform phase of the ultrasonic sample were separated and then subjected to rotary evaporation using a rotary evaporator (IKA, Staufen, Germany) to a viscous substance [9]. The chloroform and methanol (analytical grade) were purchased from Merck KGaA, Darmstadt, Germany.

Antimicrobial Susceptibility Assays
Antimicrobial susceptibility assays were performed according to a method described in our recent studies [9,10]. Blank disks (6 mm, Oxoid, Basingstoke, UK) and Mueller-Hinton (MH) were purchased from Oxoid, Basingstoke, UK. An aliquot of 10 µL crude extract (500 µg/mL) was added onto each disk and the bacteriostatic effect on the corresponding strains evaluated by measuring the diameter of the inhibition zone after incubation at 37 • C for 12 h. A gentamicin disk (10 µg, Oxoid, Basingstoke, UK) was used as a positive control, while the methanol phase with water and chloroform phase with anhydrous ethanol were used as negative controls.
Broth dilution testing (microdilution) was carried out to determine MICs of the extracts according to the standard method issued by the Clinical and Laboratory Standards Institute, USA (CLSI, M100-S28, 2018). The standard solution of gentamicin (100 µg/mL) was purchased from the National Standard Material Information Center, Beijing, China [9].

Prep-HPLC Analysis
The Prep-HPLC analysis was performed as described in our recent studies [9,10]. The extract sample (10 mg/mL) was dissolved in ultrapure water (analytical grade, Merck KGaA, Darmstadt, Germany), centrifugated at 8000× g for 20 min, and then passed through 0.22 µm membrane (Shanghai Sangon Biological Engineeing Technology and Service Co., Funding: This work was supported by the Shanghai Municipal Science and Technology Commission, grant 17050502200, and the National Natural Science Foundation of China, grant 31671946.